An autoimmune disease or disorder occurs when the body's immune system attacks and destroys healthy body tissue by mistake. Autoimmune diseases can attack almost any tissue in the body and all autoimmune diseases are characterized by local inflammation and infiltration by immune cells called lymphocytes.
As an example, autoimmune diabetes, also known as Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM), occurs when the body's immune system mistakenly destroys the pancreatic cells, called beta cells, that make insulin. Damage to beta cells results in an absence or insufficient production of insulin produced by the body. In all autoimmune diseases, including autoimmune diabetes, lymphocytes migrate from the blood stream into target tissues via interactions with the extracellular matrix (ECM). In the case of autoimmune diabetes, lymphocytes attack pancreatic islets via interaction with ECM that lies between islet capillaries and endocrine cells.
One in three hundred American children will develop autoimmune diabetes. Many of these individuals can be identified before they present with hyperglycemia through screening for autoimmune diabetes associated autoantibodies. Thus, there is a therapeutic window where autoimmune diabetes could be prevented, given the knowledge and means to do so. The present application describes novel strategies to reverse and/or prevent the progression to autoimmune diabetes in at-risk individuals.
As another example, multiple sclerosis (MS) is also an autoimmune disease but in MS the autoimmune activity is directed against central nervous system (CNS) antigens. The disease is characterized by inflammation in parts of the CNS, leading to the loss of the myelin sheathing around neuronal axons (demyelination), axonal loss, and the eventual death of neurons, oligodendrocytes and glial cells. For a comprehensive review of MS and current therapies, see, e.g., Compston, A., et al., McAlpine's Multiple Sclerosis 4th ed., Churchill Livingstone Elsevier (2006).
MS is one of the most common diseases of the CNS in young adults, and an estimated 2.5 million people suffer from MS. MS is a chronic, progressing, disabling disease, which generally strikes its victims sometime after adolescence, with diagnosis generally made between 20 and 40 years of age, although onset can occur earlier. The disease is not directly hereditary, although genetic susceptibility plays a part in its development. MS is a complex disease with heterogeneous clinical, pathological and immunological phenotype.
There are four major clinical types of MS: 1) relapsing-remitting MS (RRMS), characterized by clearly defined relapses with full recovery or with sequelae and residual deficit upon recovery; periods between disease relapses are characterized by a lack of disease progression; 2) secondary progressive MS (SPMS), characterized by an initial relapsing remitting course followed by progression with or without occasional relapses, minor remissions, and plateaus; 3) primary progressive MS (PPMS), characterized by disease progression from onset with occasional plateaus and temporary minor improvements allowed; and 4) progressive relapsing MS (PRMS), characterized by progressive disease onset, with clear acute relapses, with or without full recovery; periods between relapses characterized by continuing progression.
Clinically, the illness most often presents as a relapsing-remitting disease and, to a lesser extent, as steady progression of neurological disability. Relapsing-remitting MS presents in the form of recurrent attacks of focal or multifocal neurologic dysfunction. Attacks can occur, remit, and recur, seemingly randomly over many years. Remission is often incomplete and as one attack follows another, a stepwise downward progression ensues with increasing permanent neurological deficit. The usual course of RRMS is characterized by repeated relapses associated, for the majority of patients, with the eventual onset of disease progression. The subsequent course of the disease is unpredictable, although most patients with a relapsing-remitting disease will eventually develop secondary progressive disease. In the relapsing-remitting phase, relapses alternate with periods of clinical inactivity and may or may not be marked by sequelae depending on the presence of neurological deficits between episodes. Periods between relapses during the relapsing-remitting phase are clinically stable. On the other hand, patients with progressive MS exhibit a steady increase in deficits as defined above and either from onset or after a period of episodes, but this designation does not preclude the further occurrence of new relapses.
In healthy individuals (i.e., those without an autoimmune disease or disorder), immune tolerance is maintained by populations of regulatory T-cells including FoxP3+ regulatory T-cells (Treg) (Sakaguchi, S., et al., Nat. Rev. Immunol. 10, 490-500 (2010)). Treg absence or depletion leads to multi-systemic autoimmunity in mice and humans (Wildin, R. S., et al., Nat. Genet. 27, 18-20 (2001)) whereas adoptive transfer of Treg can abrogate autoimmunity.
In MS, Treg present in the CNS are known to limit the extent of neuroinflammation and to facilitate clinical recovery during the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), such that multiple investigative therapeutic strategies to treat autoimmune demyelination are directed at promoting the number and/or function of Treg. However, existing therapies have not managed to induce stable, functional FoxP3+ Treg, in part because Treg in vivo are a population in flux. Natural Treg (nTreg) continually emerge through thymic selection, whereas induced Treg (iTreg) originate in peripheral tissues in response to inflammatory stimuli and can revert into effector T-cells. This variability in the number and function of local Treg at sites of inflammation can impact the durability of immune tolerance in peripheral tissues.
Despite the fact that the inflammatory milieu is known to have decisive effects on immune tolerance, little is known about how the tissue micro-environment influences the function and number of Treg. Therefore, there is increasing interest in the role of ECM at the interface between lymphocytes and local cells in autoimmunity (Bollyky, P. L., et al., Curr. Diab. Rep. 12, 471-480 (2012); Irving-Rodgers, H. F., et al., Diabetologia 51, 1680-1688 (2008); Hull, R. L., et al., J. Biol. Chem. 287, 37154-37164 (2012); Bitan, M., et al., Diabetes. Metab. Res. Rev. 24, 413-421 (2008); Ziolkowski, A. F., et al., J. Clin. Invest. 122, 132-141 (2012)).
One tissue component that is abundant at sites of inflammation is hyaluronan (HA), an extracellular matrix (ECM) polysaccharide. HA has many functions, such as providing support and anchorage for cells, segregating tissues from one another, facilitating cell to cell signaling, development, migration and function (Bollyky, P. L., et al. (2012), supra). HA is synthesized by a class of integral membrane proteins called hyaluronan synthases and extruded through the cell membrane into the extracellular space (Laurent, T. C., et al., Immunol. Cell Biol. 74, A1-7 (1996)).
HA is a polymer of disaccharides composed of glucuronic acid and N-acetylglucosamine and linked via alternating β-1,4 and β-1,3 glycosidic bonds. HA can be 25,000 disaccharide repeats in length. In vivo polymers of HA can range in size from 5,000 to 20,000,000 Da. HA is synthesized by a class of integral membrane proteins called hyaluronan synthases, of which vertebrates have three types: HAS1, HAS2, and HAS3. These enzymes lengthen hyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide as it is extruded through the cell membrane into the extracellular space.
HA is a key mediator of inflammation, with roles in lymphocyte trafficking, proliferation, and antigen presentation (Laurent, T. C., and Fraser, J. R., FASEB J. 6, 2397-2404 (1992); Bollyky, P. L., et al., Cell Mol Immunol. 3, 211-220 (2010)). HA is increased in lesions associated with human autoimmune diseases, including multiple sclerosis, Sjögrens disease, and autoimmune thyroiditis (Back, S. A., et al., Nat. Med. 11, 966-972 (2005); Engström-Laurent, A. “Changes in hyaluronan concentration in tissues and body fluids in disease states.” The Biology of Hyaluronan, CIBA Foundation Symposium, 143, 233-47 (1989); Gianoukakis, A., et al., Endocrinology 148, 54-62 (2007). HA is also increased in the serum of individuals with Lupus, rheumatoid arthritis, psoriasis, and autoimmune thyroiditis (Engström-Laurent, supra; Pitsillides et al., Rheumatol. 33, 5-10 (1994); Hansen, C., et al., Clin. Exp. Rheumatol. 14 Suppl. 15, S59-67 (1996); Torsteinsdottir et al., Clin. Exp. Immunol. 115, 554-560 (1999); Elkayam, O., et al., Clin. Rheumatol. 19, 455-457 (2000); Kubo, M., et al., Arch. Dermatol. Res. 290, 579-581 (1998).
HA is highly abundant within chronically inflamed tissues, including for example MS lesions (Bollyky, P. L., et al. (2012), supra; Back, S. A., et al., supra). For example, in one study HA was shown to accumulate in demyelinated lesions in MS and EAE. Immunostaining for PLP of a chronic MS lesion showed complete loss of myelin in the center of the lesions. CD44 staining revealed high levels of CD44 in the lesions, and elevated CD44 expression in GFAP-expressing reactive astrocytes were also found. HA staining showed high levels of HA in demyelinated regions of the lesions but at lower levels in the lesion borders (Back S. A., et al., supra).
Typically, HA present within chronically inflamed tissues takes the form of short, highly catabolized fragments (as reviewed in Bollyky, P. L., et al. (2012), supra) that are pro-inflammatory agonists of Toll-like receptor signalling (Laurent, T. C., et al., Immunol. Cell Biol. 74, A1-7 (1996); Jiang, D., et al., Physiol. Rev. 91, 221-264 (2011)), driving dendritic cell maturation, and promoting phagocytosis (Jiang, D., et al., Nat. Med. 11, 1173-1179 (2005); Termeer, C., et al., J. Exp. Med. 195, 99-111 (2002)). HA overexpression tends to drive inflammation (Olsson, M. et al., PLoS Genet. 7, e1001332 (2011)), presumably through production of increased HA fragments, while inhibition of HA synthesis, including treatment with 4-methylumbelliferone (4-MU, Hymecromone), tends to reduce inflammation (Yoshioka, Y., et al., Arthritis Rheum. 65, 1160-1170 (2013); McKallip, R. J., et al., Toxins (Basel) 5, 1814-1826 (2013); Colombaro, V. et al., Nephrol. Dial. Transplant 28, 2484-2493 (2013); Saito, T., et al., Oncol. Lett. 5, 1068-1074 (2013)). With respect to the role of HA in local immune modulation, it is known that low molecular weight HA (LMW-HA) fragments inhibit the function of FoxP3+ Treg (Bollyky, P. L., et al., J. Immunol. 179, 744-747 (2007); Bollyky, P. L., et al., J. Immunol. 183, 2232-2241 (2009)). These effects are mediated via TLR signaling and via interactions with the HA receptor CD44.
In the healthy CNS, astrocytes are the main producers of low levels of HA, depositing it as ECM complexes in the spaces between myelinated axons and between myelin sheaths and astrocyte processes (Asher, R., et al., J. Neurosci. Res. 28, 410-421 (1991)). Upon injury, however, reactive astrocytes produce abundant amounts of HA, which accumulate in damaged areas (Back, S. A., et al., supra; Struve, J., et al., Glia 52, 16-24 (2005); Bugiani, M., et al., Brain 136, 209-222 (2013)). As such, HA is present at high levels in demyelinating lesions in MS patients and in mice with EAE (Back, S. A., et al., supra).
4-MU is a selective inhibitor of HA synthesis. The compound was first used in vitro in 1995 by Nakamura et al., to inhibit HA-synthesis in skin fibroblasts. Nakamura, T., et al., Biochem. Biophys. Res. Commun. 208, 470-475 (1995). In 2004, the mechanism of 4-MU was discovered by Kakizaki et al., and since then it has been used in in vivo studies in mice and rats to investigate the 4-MU influence, mainly in cancer studies (Kakizaki, I., et al., J. Biol. Chem. 279, 33281-33289 (2004); see also, e.g., Yoshihara, S., et al., FEBS Letters 579, 2722-2726 (2005); Lokeshwar, V. B., et al., Cancer Res. 70, 2613-2623 (2010)) and in atherosclerosis studies (Nagy, N., et al., Circulation 122, 2313-2322 (2010)). 4-MU is also already used in humans. It is available without a prescription as Heparvit, a nutraceutical product for cancer patients. Furthermore, it is available with prescription in Europe and Asia to treat biliary spasm under the name Hymecromone. In that setting, the drug has an excellent safety profile and has been used for several years.
Although it is known that HA deposits are abundant in chronically inflamed tissues and that 4-MU is a selective inhibitor of HA synthesis, there remains a need to develop a safe and effective therapy for autoimmune diseases and disorders such as, for example, diabetes and MS, by providing a well-founded understanding of the role of HA in autoimmune pathogenesis.